Project 1. Defining upstream and downstream components of Nodal signaling in gastrulation.[unreadable] [unreadable] Modulators of sqt penetrance. Background: cyc embryos have a more limited mesoderm and endoderm deficit than sqt;cyc double-mutant embryos, namely a reduction in anterior axial mesoderm and endoderm, leading to holoprosencephaly (HPE). This phenotype is seen in only a fraction of sqt mutants, with many appearing wild type. At NHGRI, we previously examined the basis of sqt phenotypic variability and found environmental and genetic factors that influence HPE incidence. This year we extended our analysis to a novel sqt phenotype - bifurcation at the midline and find that the same genetic and environmental risk factors for HPE increase increase midline bifurcation, but that perturbations in WNT signaling specifically increase midline bifurcation incidence without afffecting HPE. [unreadable] [unreadable] Project 2. Identifying roles of RhoGTPase signaling in gastrulation movements.[unreadable] Background: RhoGEF proteins are positive regulators of RhoGTPases, which have profound roles in cellular movement and morphology. To identify RhoGEFs with roles in directing the morphogenetic events of gastrulation, we performed a loss-of-function screen. We identified 48 RhoGEFs expressed during early embryogenesis and determined the loss-of-function phenotypes for 23 of these, using a non-invasive embryo holding system we designed that allows for the parallel time-lapse documention of 54 embryos. We thus identified five RhoGEFs for which two independent MOs produced the same phenotype. Three of these, homologues of ARHGEF16, Frabin and Net1, respectively, disrupted epiboly. Two others, ARHGEF10 and PLEKHG4 homologues, caused post-gastrulation defects during somitogenesis stages. We are now engaged in control experiments to confirm the specificity of these phenotypes, with a focus on the epiboly mutants. [unreadable] [unreadable] Project 3. Defining roles of blastula margin-enriched proteins and behaviors of blastula margin cells.[unreadable] Background: A precise separation of newly-specified germ layer precursors in zebrafish embryos, has been untenable using conventional tools. Over previous funding years we have developed and refined a novel technique for precisely microdissecting embryonic regions of interest: FACS-assisted microdissection of photolabeled cells (FAM-P). We used this method to separate mesoderm and endoderm (mesendoderm) precursor cells from ectoderm precursor cells of pre-gastrula stage embryos, and used microarrays to assess their respective transcriptomes. Over the last 12 months, we have compared the behaviors of mesendoderm and ectoderm precursors in transplantation assays and in cell cycle assays. This has shown us that both populations retain their documented properties, and has revealed cell cycle differences between these two populations. We also performed a detailed analysis of the microarray data from the previous funding period, revealing that mesendoderm precursor cells express fewer enzyme-encoding genes and demonstrating that FAM-P purification effectively depletes extra-embryonic cells. Finally, we have investigated the requirements of several mesendoderm precursor-specific genes identified in these studies, using antisense reagents. One of these, dusp4, is required for normal head development and the differentiation of late endoderm. Our manuscript describing this work is currently in press at PNAS. We are continuing to study dusp4 function, particularly focusing on its role as an upstream activator pf p53, which we discovered. We are also looking at the function of the transcription factor Six4.2, whose loss of function we find affects the positioning of late endoderm. Finally, we are examining the expression and function of a large set of genes expressed in extra-embryonic tissues adjacent to the mesendoderm, in hopes of identifying signals that initiate mesendoderm development.